Plasmids can provide advantageous traits to host bacteria, although they may impose a fitness cost. Chromosome-encoded factors are important for regulating the expression of genes on plasmids, and host chromosomes may differ in terms of their interactions with a given plasmid. Accordingly, differences in fitness cost loading and compensatory co-evolution may occur for various host chromosome/plasmid combinations. However, the mechanisms of compensatory evolution are highly divergent and require further insights. Here, we reveal novel evolutionally mechanisms of Pseudomonas putida KT2440 to improve the fitness cost imposed by the incompatibility P-1 (IncP-1) multidrug resistance plasmid RP4. A mixed culture of RP4-harboring and-free KT2440 cells was serially transferred every 24 h under non-selective conditions. Initially, the proportion of RP4-harboring cells decreased rapidly, but it immediately recovered, suggesting that the fitness of RP4-harboring strains improved during cultivation. Largersized colonies appeared during 144-h mixed culture, and evolved strains isolated from larger-sized colonies showed higher growth rates and fitness than those of the ancestral strain. Whole-genome sequencing revealed that evolved strains had one of two mutations in the same intergenic region of the chromosome. Based on the research of another group, this region is predicted to contain a stress-inducible small RNA (sRNA). Identification of the transcriptional start site in this sRNA indicated that one mutation occurred within the sRNA region, whereas the other was in its promoter region. Quantitative reverse-transcription PCR showed that the expression of this sRNA was strongly induced by RP4 carriage in the ancestral strain but repressed in the evolved strains. When the sRNA region was overexpressed in the RP4-free strain, the fitness decreased, and the colony size became smaller. Using transcriptome analysis, we also showed that the genes involved in amino acid metabolism and stress responses were
H-NS family proteins regulate the expression of many genes by preferably binding to AT-rich genomic regions and altering DNA topology. They are found in both bacterial chromosomes and plasmids, and plasmid-encoded H-NS family proteins have sometimes been suggested to act as a molecular backup of the chromosomally encoded ones. Pmr is an H-NS family protein encoded on the catabolic plasmid pCAR1, which belongs to incompatibility P-7 group. We have investigated the function of Pmr in Pseudomonas putida KT2440, where two H-NS family proteins (TurA and TurB) encoded on the chromosome are expressed predominantly. Previous transcriptome analyses suggested that TurA, TurB, and Pmr cooperatively regulate numerous genes, but the differentially transcribed genes in KT2440 turA(pCAR1), KT2440 turB(pCAR1), and KT2440(pCAR1 pmr) compared with those in KT2440(pCAR1) were somewhat different. Here, we performed RNA sequencing analyses to compare the differentially transcribed genes after the deletion of turA or turB in KT2440, and turA, turB or pmr in KT2440(pCAR1). Three pCAR1-free strains (KT2440, KT2440 turA, KT2440 turB) and four pCAR1harboring strains [KT2440(pCAR1), KT2440 turA(pCAR1), KT2440 turB(pCAR1), KT2440(pCAR1 pmr)], grown until the log and stationary phases, were used. In KT2440, TurA was the major H-NS family protein regulating a large number and wide range of genes, and both TurA and TurB were suggested to functionally compensate each other, particularly during the stationary phase. In KT2440(pCAR1), the numbers of differentially transcribed genes after the deletion of turA or turB drastically increased compared to those in KT2440. Notably, more than half of the differentially transcribed genes in KT2440 turA and KT2440 turB did not overlap with those in KT2440 turA(pCAR1) and KT2440 turB(pCAR1). This dynamic change could be explained by the acquisition of pCAR1 itself and the expression of Pmr. After pCAR1
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